Comprehensive insights on genetic alterations and immunotherapy prognosis in Chinese melanoma patients

Immune checkpoint inhibitors (ICI) have emerged as a promising therapeutic option for melanoma, which demonstrating improved clinical outcomes in melanoma patients regardless of specific genetic mutations. However, the identification of reliable biomarkers for predicting immunotherapy response and prognosis remains a challenge. In this study, we performed genetic profiling of the melanoma patients with different subtypes and evaluated the efficacy of ICI treatment. A total of 221 melanoma patients were included in our cohort, consisting primarily of acral lentiginous melanoma (ALM), cutaneous malignant melanoma (CMM), and mucosal malignant melanoma (MMM). Genetic analysis revealed BRAF mutations was predominant in CMM and NRAS mutations was prevalent in ALM. Copy number variants (CNVs) and structural variants (SV) were also detected, with CCND1 and CDK4 being the most affected genes in CNV and BRAF, ALK and RAF1 being the druggable targets in SV. Furthermore, NRAS mutations were associated with a poor prognosis in ALM, while TERT mutations were linked to unfavorable outcomes in CMM after receiving PD-1 therapy. Additionally, ALK expression exhibited improved outcomes in both ALM and CMM subtypes. Our study provides a comprehensive genomic and pathological profiling of Chinese melanoma patients, shedding light on the molecular landscape of the disease. Furthermore, numbers of gene mutations and ALK expression were identified as prognostic indicators. These findings contribute to the understanding of melanoma genetics in the Chinese population and have implications for personalized treatment approaches.


Pathological diagnosis
The pathological features of patients were identified based on the NCCN guideline criteria 20 .ALM, CMM and MMM were diagnosed based on the site of disease.ALM appears on the palms of the hands, soles of the feet, or under the nails.CMM refers to melanoma that occurs on the skin's surface.It often develops in areas exposed to the sun, such as the face, arms, legs, and back.MMM occurs in the mucous membranes lining areas like the mouth, nose, throat, vagina, or anus.

NGS assay
Between 10 and 500 ng of TNA was used as an input template for NGS library construction beginning with an RT reaction that converted RNA to cDNA without altering carryover gDNA.Input template was mixed with random hexamers and heated to 65 ℃ for 5 min, and immediately chilled in icy water.Reverse transcriptase,RNase inhibitor, and dNTPs (Enzymatics) were added and incubated at 42 ℃ for 2 h, followed by 70 ℃ for 15 min.The second strand was synthesized with Escherichia coli DNA polymerase I and RNase H (Enzymatics) at 16 ℃ for 1 h.After SPRI beads cleanup (Beckman), DNA were enzymatically fragmented by DNA fragmentation enzyme mix (New England Biolabs or Kapa Biosystems) and end-polished by T4 DNA polymerase, Taq polymerase, and T4 polynucleotide kinase (Enzymatics).This polished fragment is ligated with a set of adaptors.Each adaptor consisted of a designated P5 index for sample deconvolution and a degenerate unique molecular identifier (UMI) for amplicon binning.Ligated templates were SPRI-cleaned and mixed with the first pool of target-specific primers and thermal stable DNA polymerase (Thermo Fisher), and subjected to multiple linear amplification cycles, each producing a complementary strand copy of the input template.All primers share a common 5 tag suppressing amplification between genespecific primers owing to hairpin formation in such amplicons.After cleanup, a second pool of target-specific primers nested to the first pool, a common adaptor primer, and an indexed P7 primer were applied for PCR amplification.Resulting libraries were pooled and sequenced using HiSeq 10 or

NGS data processing
Raw data were demultiplexed using Illumina bcl2fq version 2.19 for P7 index, followed by a custom script for matched P5 index demultiplexing, BBduk for adaptor trimming, and UMI parsing.Fastq sequences were aligned to human reference genome (hg19) using BWA MEM with the default setting.On-target alignments were extracted using BedTools 2.27 21 supplied with specific panel bed files.SNVs were called using a UMI-aware custom script that includes samtools and bcftools.SNVs with occurrence frequency higher than 1% in the dbSNP database were regarded as polymorphisms and filtered.Gene fusions were called based on BWA MEM supplementary alignments, taking into account different mapping start positions, and the breakpoint frame status was inferred based on RefSeq open reading frames.We favored BWA MEM over common RNA-seq aligners such as STAR, TOPHAT, or Mapsplice 22 because its algorithm was advantageous for fusions involving 2 different genes.BWA MEM also allowed for simultaneous gDNA (resulting bam file is compatible with the mutation calling module) and cDNA read alignment, which was unique to our wet laboratory method.

Statistical analysis
Survival data were analyzed with the Kaplan-Meier method, and log-rank tests were used for comparisons between different groups.Cox proportional hazards regression analysis was conducted to identify the following possible predictors of RFS in melanoma patients: NRAS status (positive vs negative), TERT status (positive vs negative), ALK mRNA status (positive vs negative), BRAF status (positive vs negative), KIT status (positive vs negative), KRAS status (positive vs negative) and TP53 status (positive vs negative).All statistical analyses were conducted using GraphPad Prism (version 8.02) software.The results are expressed as the mean ± SEM.The data were analyzed using a t test or one-way ANOVA, followed by the appropriate post-hoc tests (GraphPad Prism 7, GraphPad, CA, USA).

Ethical approval and consent to participate
The study was approved by Ethics Committee of the Zhejiang Cancer Hospital (IRB-2021-301), and informed consent was obtained from all subjects.

Patient characteristics and genetic alterations
A total of 221 melanoma patients were included in the cohort (Table 1).A significant difference was found in genders, with the number of males being twice as high as females (Table 1, 63% vs 37%, P < 0.05).The age distribution ranged from 20 to 90 years, with an average age of 62 years for the cohort.
To evaluate the association between genes, co-occurrence and mutual exclusivity analyses were conducted for the highly frequent genes (Fig. 1B).CNVs showed a high frequency of co-occurrence (Fig. 1B).For example, CCND1 had a significant co-occurrence with CDK4, FGF3/4/19 (P < 0.05).Additionally, sporadic SNVs were also found in co-occurring genes.Co-occurrence was observed between BRAF and ERBB4, PTEN.Mutual exclusivity was mainly observed with BRAF, for instance, it was mutually exclusive with CCND1, FGF19, KIT, NF1, and NRAS.

Clinicopathologic and molecular features difference between melanoma subtypes
The pathologic diagnosis was conducted and 3 different pathological subtypes were identified (Table 1), including ALM (n = 126, 57%), CMM (n = 81, 36.7%) and MMM (n = 14, 6.3%).Obviously, ALM accounted a significant proportion in the cohort, which was 9 times higher than MMM.The location of symptoms was diverse in each subtype (Table 1).In the ALM group, feet (n = 109, 86.5%) was the dominate site, followed by hands (n = 16, 12.7%).Compared to the ALM, the site of onset was more diverse in CMM.Leg (n = 22, 27%) was the primary site in CMM.Abdomen (n = 16, 20%) and back (n = 12, 15%) were found to be the second and third location.

A P C A S C L 2 B R A F C D K N 2A C T N N B 1 E R B B 4 FA T 3 F G F 19 F G F 3 F O X L 2 G N A S H R A S K IT K R A S M D M 2 M Y C N F 1 N R A S P R P 1B P T E N R A F 1 R E L A R IC T O R T E R T T P 53 C C N D 1 C D K 4 F G F 4
A B www.nature.com/scientificreports/ In the MMM group, head and neck was the frequent site at diagnosis.In order to describe the clinicopathologic features between subtypes.The age at diagnosis was analyzed and the significant difference was found between ALM and CMM, which demonstrated the higher age in ALM group (Fig. 2A, P < 0.01).Moreover, a significant difference in gender distribution was found in ALM, the number of male (n = 89) was much higher than female (n = 39) (Fig. 2B, P < 0.01).
In order to identify genetic distribution difference between melanoma subtypes, the sequencing results were further analyzed based on subtypes.The number of high frequent genes was similar between ALM (n = 28) and CMM (n = 29), which was twice higher than MMM (n = 13).Although the gene number was similar between ALM and CMM, the genetic patterns were different (Fig. 2C).The top 3 genes with SNVs in ALM were NRAS (n = 34, 31%), NF1 (n = 17, 16%) and BRAF (n = 15, 14%).In the CMM group, BRAF (n = 43, 61%) was the most frequent gene, which was much higher than TP53 (n = 11, 15%) and CDKN2A (n = 10, 14%).The genetic distribution pattern in MMM was similar to ALM, NRAS (n = 4, 31%) carried the highest proportion, followed by BRAF (n = 3, 23%) and FAT3 (n = 2, 15%).Different from SNV, the proportion of top3 genes with CNVs was similar.CCND1 was the primary gene in both ALM and CMM, followed by CDK4 and FGF19 in ALM, MYC and HRAS in CMM.In MMM, CDK4, FOXL2 and MDM2 shared same proportion (Fig. 2D).The number of oncogenic driver genes in fusions was less than SNV and CNV (Fig. 2E).Totally, only 5 driver genes were identified in the cohort.ALM and CMM carried 4 of 5, respectively.In ALM, BRAF and TERT had the highest proportion followed by ALK and RELA.Similarly, besides RAF1, TERT, BRAF and ALK were also found in CMM.There was no fusions in MMM.

Characteristics of patients treated with immune checkpoint inhibitors
In total, 51 patients in the cohort received ICIs treatment (Fig. 3).The number of patients with ALK, CMM and MMM were 33, 17 and 1, respectively.Seven patients did not reach disease progress from ICIs treatment still now.The average length of relapse free survival (RFS) of those 7 patients was 41.2 months.Among them, the longest RFS was 53.6 months, followed by 48.2 months, 45.2 months, 43.8 months, 42.3 months, 33.5 months and 21.9 months.In the patients who were found to be disease progress (n = 44), the RFS length was variable, which was between 0.7 years to 35.6 months.Among them, the RFS of 38 patients were less than 12 months.

Clinical outcomes of PD-1 therapy in patients with different gene mutations
The association between gene mutations and prognostic outcomes were further analyzed.Firstly, the highly frequent genes in the cohort were included in the analysis.There was no significant difference in prognostic outcomes between BRAF, KIT, KRAS and TP53 mutant and wild groups (Fig. S1A-D).On the contrary, NRAS was found to be highly related to poor prognosis (Fig. 4A, HR, 1.877, 95% CI 0.8958 to 3.935; P < 0.05).Furthermore, the effect of clinicopathologic subtypes on prognostic outcomes was further explored.However, there was no significant difference was found between ALM and CMM (Fig. S1E).Interestingly, the particular gene mutation in ALM and CMM was significantly related to the prognosis.In the ALM, the RFS for NRAS mutant was significant lower than wild type group (Fig. 4B, HR 2.347, 95% CI 0.9542 to 5.772; P < 0.05).CMM displayed a different pattern, the significant difference in prognosis was found in TERT (Fig. 4C, HR 4.108, 95% CI 0.8072 to 20.91; P < 0.01).

Discussion
Melanoma is renowned for its extensive genetic heterogeneity, with various genetic mutations driving its initiation, progression, and resistance to therapy 23 .The identification and understanding of these genetic mutations hold significant importance for subsequent therapeutic interventions, including immunotherapy.In this study, we aimed to comprehensively profile the genetic landscape of Chinese melanoma patients with different subtypes.Our analysis revealed numerous oncogenes and tumor suppressors affected by SNVs, such as BRAF, NRAS, and NF1.Notably, the BRAF V600E mutation accounted for a substantial proportion of BRAF variations.Furthermore, we identified CCND1, CDK4, and FGF9 as the top three genes with the highest frequency of CNVs.Additionally, we discovered several oncogenic drivers associated with rearrangements, including BRAF, RAF1, and ALK.In addition to characterizing the genetic landscape, we explored the association between genetic mutations and the outcomes of immunotherapy.Our findings indicated a negative prognosis associated with NRAS mutations in ALM.Similarly, we observed unfavorable prognostic outcomes in CMM patients with TERT mutations.Moreover, we identified a relationship between ALK mRNA levels and immunotherapy efficacy, as patients with ALK expression exhibited improved prognostic outcomes in both ALM and CMM.
In contrast to the high incidence of Caucasian melanoma, melanoma morbidity is lower in the Asian population 24,25 .This disparity in incidence highlights the extensive findings that can be drawn from melanoma studies conducted on Caucasian populations.However, research on melanoma in the Asian population remains limited due to its lower occurrence.Previous studies have shown that CMM (> 90%) is more common among Caucasian populations, particularly those with fair skin and a high susceptibility to ultraviolet (UV) radiation 26 .On the contrary, ALM is prevalent among Asian populations, as reported in previous Asian studies 27 .In our   www.nature.com/scientificreports/cohort, ALM accounted for the majority (57.4%), followed by CMM (36%) and MMM (6.6%).In terms of somatic gene mutations, the mutation rates of BRAF, NRAS, NF1, KIT, and TERT were 26, 19, 10, 8, and 4%, respectively.The mutation rates of BRAF, NF1, and TERT were like those reported in previous studies on Asian populations 28 .However, the NRAS mutations in our cohort was almost twice higher than the proportion in other studies (19% vs 10%).This difference may be attributed to the significant number of NRAS-mutant patients in our specific cohort or the prevalence of synonymous mutations in this cohort.In both our cohort and other studies 28,29 , BRAF mutations were predominantly V600 hotspot mutations, particularly V600E, which were mainly observed in CMM.However, unlike previous studies that demonstrated a higher proportion of BRAF non-V600E mutations MMM, our cohort showed that BRAF non-V600E mutations were also dominant in CMM.This variation could be attributed to the substantial difference in the proportion of MMM between the two cohorts.

N R A S N F 1 B R A F B R A F T P 5 3 C D K N 2 A N R A S B R A F F A
Immunotherapy has emerged as a standard treatment option for melanoma patients due to the limited availability of targetable driver mutations.Previous studies have shown that immunotherapy can provide benefits to melanoma patients, but the prognostic outcomes vary among cohorts with different gene mutations.Douglas et al. demonstrated that the overall response rate (ORR) in melanoma patients (n = 11) with positive NRAS mutations was significantly higher (64% vs 30%) than those with wildtype NRAS (n = 33) after receiving immunotherapy 30 .However, Michael et al. suggested the similar ORR in both NRAS mutant and wildtype melanoma patients 31 .These results indicate that the impact of NRAS mutations on the prognosis of immunotherapy still needs further exploration.Furthermore, since CMM was the dominant subtype in both Caucasian cohorts, these results may not reflect the immunotherapy outcomes in Asian cohorts due to ethnic and pathological differences.The Asian study conducted by Zhou et al. included both CMM (n = 92) and non-CMM (n = 114) subtypes in the cohort, demonstrating that the response rates in patients with NRAS mutations from both CMM and non-CMM groups were significantly lower than those without NRAS mutations 16 .However, this pooled analysis included three different PD-1 inhibitors (toripalimab, pembrolizumab, SHR-1210), which may have led to biased conclusions.Additionally, the predominant pathological subtype in Asian melanoma patients, ALM, was not further analyzed in the non-CMM group.In our study, we analyzed the immunotherapy outcomes in CMM, ALM, and MMM subtypes after administering only one PD-1 inhibitor (pembrolizumab).In contrast to the above-mentioned study, the response rates showed similar results in both NRAS mutant and wildtype groups in CMM.However, patients with NRAS wildtype showed a better response than those with NRAS mutations in ALM.The variation in prognosis outcomes in CMM between our study and Zhou et al. 's study may attribute to differences in the cohorts.The aforementioned Asian study included melanoma patients from four clinical www.nature.com/scientificreports/trials who received different PD-1 inhibitors.The diversity in patient inclusion criteria and complex treatment strategies across different clinical trials could affect the conclusions drawn.
In addition to SNVs, recent studies have discovered that the expression of ALK is associated with the efficacy of immunotherapy in melanoma.A previous study demonstrated a prevalence of ALK expression of 3% (16/603) in CMM 32 .However, in our study, the rates of ALK expression in CMM and ALM were 41% and 42%, respectively.Such a significant difference can be attributed to variations in the specificity of the screening tools used in both cohorts.The previous study and our study employed immunohistochemistry (IHC) and RNA sequencing (RNAseq) to detect ALK expression, respectively.Previous research has shown that the specificity of IHC is only 52.2% 33 .Furthermore, IHC specificity is highly dependent on the type of antibody used and the cutoff determined by the operators.Therefore, compared to IHC, RNAseq can more accurately reflect the true proportion of ALK expression-positive patients in melanoma.Co-occurrence can be observed between SNVs and ALK expression.Previous research demonstrated a high prevalence of ALK expression in melanoma patients with wildtype BRAF or NRAS 34,35 .The same phenomenon was confirmed in our study.Furthermore, studies have found an association between ALK expression and prognostic outcomes.A previous study showed that ALK expression has a favorable impact on the prognosis of NRAS-mutated melanomas 36 .Additionally, the status of BRAF and KRAS did not have a significant relation to prognostic outcomes in patients with ALK expression.Similar to the previous study, we did not find an association between ALK expression and prognosis in the KRAS mutant group.On the contrary, ALK expression had a favorable impact on the prognosis of NRAS wild type melanomas.Furthermore, in the BRAF mutant group, better prognostic outcomes were observed in patients with positive ALK expression.We speculate that the different results between our study and the previous study are due to differences in the screening tool and cohort size.The aforementioned study employed IHC to detect ALK expression, which may have underestimated the number of patients with ALK expression.Additionally, only 22 patients were included in the cohort, which could have resulted in biased results.In addition to the co-occurrence between SNVs and ALK expression, our study also demonstrated that ALK expression alone can negatively impact the prognosis of melanoma patients with both CMM and ALM.This suggests that ALK expression alone can serve as an indicator to predict immunotherapy efficacy.
Several limitations should be acknowledged in our study.Information on patient characteristics, such as stages, primary or metastatic status, was missing.This information could provide a more comprehensive exploration of the association between genomic mutations and clinicopathological features.Additionally, https://doi.org/10.1038/s41598-024-65065-6www.nature.com/scientificreports/

Figure 4 .
Figure 4.The Kaplan-Meier curves for RFS in patients with SNVs after PD-1 therapy in melanoma.(A) NRAS mutation is the negative prognostic indicator of PD-1 therapy in melanoma without distinguishing pathological subtypes.(B) NRAS mutation is the negative prognostic indicator of PD-1 therapy in ALM.(C) TERT mutation is the negative prognostic indicator of PD-1 therapy in CMM.SNV single nucleotide variant, ALM acral lentiginous melanoma, CMM cutaneous malignant melanoma.Positive, the specific gene mutation (NRAS or TERT) was detected in patients by DNA sequencing.Negative, the specific gene mutation (NRAS or TERT) was not detected in patients by DNA sequencing.RFS relapse-free survival.

Figure 5 .
Figure 5.The Kaplan-Meier curves for RFS in patients with ALK mRNA after PD-1 therapy in melanoma.(A) ALK mRNA expression is a positive prognostic indicator in melanoma without distinguishing pathological subtypes.(B) ALK mRNA expression is a positive prognostic indicator in ALM.(C) ALK mRNA expression is a positive prognostic indicator in CMM.(D) ALK mRNA expression could positively impact the prognostic outcomes of patients with BRAF mutation.(E) ALK mRNA expression could positively impact the prognostic outcomes of patients without NRAS mutation.(F) ALK mRNA expression could positively impact the prognostic outcomes of patients without KIT mutation.ALM acral lentiginous melanoma, CMM cutaneous malignant melanoma.Positive, the ALK mRNA was detected in patients by RNA sequencing.Negative, the ALK mRNA was not detected in patients by RNA sequencing.RFS relapse-free survival.

Table 1 .
Clinicopathologic features of melanoma patients.